U.S. patent application number 09/813920 was filed with the patent office on 2001-12-13 for cold-adapted equine influenza viruses.
Invention is credited to Dowling, Patricia W., Youngner, Julius S..
Application Number | 20010051161 09/813920 |
Document ID | / |
Family ID | 26889904 |
Filed Date | 2001-12-13 |
United States Patent
Application |
20010051161 |
Kind Code |
A1 |
Dowling, Patricia W. ; et
al. |
December 13, 2001 |
Cold-adapted equine influenza viruses
Abstract
The present invention provides experimentally-generated
cold-adapted equine influenza viruses, and reassortant influenza A
viruses comprising at least one genome segment of such an equine
influenza virus, wherein the equine influenza virus genome segment
confers at least one identifying phenotype of the cold-adapted
equine influenza virus, such as cold-adaptation, temperature
sensitivity, dominant interference, or attenuation. Such viruses
are formulated into therapeutic compositions to protect animals
from diseases caused by influenza A viruses, and in particular, to
protect horses from disease caused by equine influenza virus. The
present invention also includes methods to protect animals from
diseases caused by influenza A virus or other infectious agents
utilizing the claimed therapeutic compositions. Such methods
include using a therapeutic composition as a vaccine to generate a
protective immune response in an animal prior to exposure to an
infectious agent, as well as using a therapeutic composition as a
treatment for an animal that has been recently infected with an
infectious agent leading to respiratory disease, or is likely to be
subsequently exposed to such an agent in a few days whereby the
therapeutic composition reduces such respiratory disease, even in
the absence of antibody-mediated immunity. The present invention
also provides methods to produce cold-adapted equine influenza
viruses, and reassortant influenza A viruses having at least one
genome segment of an equine influenza virus generated by
cold-adaptation.
Inventors: |
Dowling, Patricia W.;
(Pittsburgh, PA) ; Youngner, Julius S.;
(Pittsburgh, PA) |
Correspondence
Address: |
HESKA CORPORATION
INTELLECTUAL PROPERTY DEPT.
1613 PROSPECT PARKWAY
FORT COLLINS
CO
80525
US
|
Family ID: |
26889904 |
Appl. No.: |
09/813920 |
Filed: |
March 21, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60194325 |
Apr 3, 2000 |
|
|
|
Current U.S.
Class: |
424/206.1 |
Current CPC
Class: |
A61K 2039/5254 20130101;
A61K 39/12 20130101; A61K 39/145 20130101; C12N 2760/16164
20130101; A61K 2039/543 20130101; C12N 7/00 20130101; C12N
2760/16134 20130101; A61K 2039/552 20130101 |
Class at
Publication: |
424/206.1 |
International
Class: |
A61K 039/145 |
Claims
What is claimed is:
1. A method to treat an animal for respiratory disease comprising
administering to said animal a therapeutic composition comprising a
virus selected from the group consisting of: (a) a cold-adapted
equine influenza virus; and (b) a reassortant influenza A virus
comprising at least one genome segment of an equine influenza virus
generated by cold-adaptation, said equine influenza virus having an
identifying phenotype selected from the group consisting of
cold-adaptation, temperature sensitivity, dominant interference,
and attenuation, wherein said equine influenza virus genome segment
confers at least one of said identifying phenotypes to said
reassortant virus.
2. The method of claim 1, wherein said respiratory disease is
caused by viral or bacterial infection or a combination
thereof.
3. The method of claim 1, wherein said respiratory disease is
caused by influenza.
4. The method of claim 1, wherein said animal is an equid.
5. The method of claim 1, wherein said therapeutic composition
comprises a cold-adapted equine influenza virus, wherein said
disease is caused by viral or bacterial infection or a combination
thereof, and wherein said therapeutic composition is administered
as a treatment to a equid, thereby eliciting a non-specific
interference response against viral or bacterial infection or a
combination thereof in said equid.
6. The method of claim 1, wherein said therapeutic composition
comprises from about 10.sup.7 TCID.sub.50 units to about 10.sup.8
TCID.sub.50 units of said virus.
7. The method of claim 1, wherein said therapeutic composition
treats an equid for respiratory disease that exhibits nasal
discharge as the major clinical sign of infection.
8. The method of claim 1, wherein said therapeutic composition
treats an equid from about 3 months to about 2 years of age against
nasal discharge.
9. The method of claim 1, wherein said therapeutic composition
treats an equid for respiratory disease in which major clinical
signs of disease are reduced within about 7 days.
10. A method to treat an animal for respiratory disease comprising
administering to said animal a therapeutic composition comprising a
virus selected from the group consisting of: (a) a cold-adapted
equine influenza virus having a dominant interference phenotype;
and (b) a reassortant influenza A virus comprising a portion of an
equine influenza virus genome conferring cold-adaptation and
dominant interference phenotypes, wherein said therapeutic
composition exhibits non-specific interference.
11. The method of claim 10, wherein said animal is an equid.
12. The method of claim 10, wherein said therapeutic composition
inhibits viral or bacterial infection, or a combination
thereof.
13. The method of claim 10, wherein said therapeutic composition
inhibits secondary viral or bacterial infection, or a combination
thereof.
14. The method of claim 10, wherein said respiratory disease is
equine influenza, and wherein said therapeutic composition exhibits
dominant interference.
15. The method of claim 10, wherein said therapeutic composition
treats an equid for respiratory disease that exhibits nasal
discharge and fever as early signs of infection.
16. The method of claim 10, wherein said therapeutic composition
treats an equid for respiratory disease that exhibits nasal
discharge as a late stage sign of infection.
17. The method of claim 10, wherein said therapeutic composition
treats an equid for respiratory disease in which major clinical
signs of disease are reduced within about 7 days.
18. The method of claim 10, wherein said therapeutic composition
treats an equid for respiratory disease in which major clinical
signs of disease are reduced within about 7 days, and race training
of the equid can resume within about 10 days of treatment.
19. The method of claim 10, wherein said therapeutic composition
comprises from about 10.sup.7 TCID.sub.50 units to about 10.sup.8
TCID.sub.50 units of said virus.
20. The method of claim 10, wherein said therapeutic composition
has an anti-viral effect.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 60/194,325, which was filed Apr. 3, 2000,
entitled "Cold-Adapted Equine Influenza Viruses".
FIELD OF THE INVENTION
[0002] The present invention relates to experimentally-generated
cold-adapted equine influenza viruses, and particularly to
cold-adapted equine influenza viruses having additional phenotypes,
such as attenuation, dominant interference, or temperature
sensitivity. The invention also includes reassortant influenza A
viruses which contain at least one genome segment from such an
equine influenza virus, such that the reassortant virus includes
certain phenotypes of the donor equine influenza virus. The
invention further includes genetically-engineered equine influenza
viruses, produced through reverse genetics, which comprise certain
identifying phenotypes of a cold-adapted equine influenza virus of
the present invention. The present invention also relates to the
use of these viruses in therapeutic compositions to protect animals
from diseases caused by influenza viruses.
BACKGROUND OF THE INVENTION
[0003] Equine influenza virus has been recognized as a major
respiratory pathogen in horses since about 1956. Disease symptoms
caused by equine influenza virus can be severe, and are often
followed by secondary bacterial infections. Two subtypes of equine
influenza virus are recognized, namely subtype-1, the prototype
being A/Equine/Prague/1/56 (H7N7), and subtype-2, the prototype
being A/Equine/Miami/1/63 (H3N8). Presently, the predominant virus
subtype is subtype-2, which has further diverged among Eurasian and
North American isolates in recent years.
[0004] The currently licensed vaccine for equine influenza is an
inactivated (killed) virus vaccine. This vaccine provides minimal,
if any, protection for horses, and can produce undesirable side
effects, for example, inflammatory reactions at the site of
injection. See, e.g., Mumford, 1987, Equine Infectious Disease IV,
207-217, and Mumford, et al., 1993, Vaccine II, 1172-1174.
Furthermore, current modalities cannot be used in young foals,
because they cannot overcome maternal immunity, and can induce
tolerance in a younger animal. Based on the severity of disease,
there remains a need for safe, effective therapeutic compositions
to protect horses against equine influenza disease.
[0005] Production of therapeutic compositions comprising
cold-adapted human influenza viruses is described, for example, in
Maassab, et al., 1960, Nature 7,612-614, and Maassab, et al., 1969,
J. Immunol. 102, 728-732. Furthermore, these researchers noted that
cold-adapted human influenza viruses, i.e., viruses that have been
adapted to grow at lower than normal temperatures, tend to have a
phenotype wherein the virus is temperature sensitive; that is, the
virus does not grow well at certain higher, non-permissive
temperatures at which the wild-type virus will grow and replicate.
Various cold-adapted human influenza A viruses, produced by
reassortment with existing cold-adapted human influenza A viruses,
have been shown to elicit good immune responses in vaccinated
individuals, and certain live attenuated cold-adapted reassortant
human influenza A viruses have proven to protect humans against
challenge with wild-type virus. See, e.g., Clements, et al., 1986,
J. Clin. Microbiol. 23, 73-76. In U.S. Pat. No. 5,149,531, by
Youngner, et al., issued Sep. 22, 1992, the inventors of the
present invention further demonstrated that certain reassortant
cold-adapted human influenza A viruses also possess a dominant
interference phenotype, i.e., they inhibit the growth of their
corresponding parental wild-type strain, as well as heterologous
influenza A viruses.
[0006] U.S. Pat. No. 4,683,137, by Coggins et al., issued Jul. 28,
1987, and U.S. Pat. No. 4,693,893, by Campbell, issued Sep. 15,
1987, disclose attenuated therapeutic compositions produced by
reassortment of wild-type equine influenza viruses with attenuated,
cold-adapted human influenza A viruses. Although these therapeutic
compositions appear to be generally safe and effective in horses,
they pose a significant danger of introducing into the environment
a virus containing both human and equine influenza genes.
SUMMARY OF THE INVENTION
[0007] The present invention provides experimentally-generated
cold-adapted equine influenza viruses, reassortant influenza A
viruses that comprise at least one genome segment of an equine
influenza virus generated by cold-adaptation such that the equine
influenza virus genome segment confers at least one identifying
phenotype of a cold-adapted equine influenza virus on the
reassortant virus, and genetically-engineered equine influenza
viruses, produced through reverse genetics, which comprise at least
one identifying phenotype of a cold-adapted equine influenza virus.
Identifying phenotypes include cold-adaptation, temperature
sensitivity, dominant interference, and attenuation. The invention
further provides a therapeutic composition to protect an animal
against disease caused by an influenza A virus, where the
therapeutic composition includes a cold-adapted equine influenza
virus a reassortant influenza A virus, or a genetically-engineered
equine influenza virus of the present invention. Also provided is a
method to protect an animal from diseases caused by an influenza A
virus which includes the administration of such a therapeutic
composition. Also provided are methods to produce a cold-adapted
equine influenza virus, and methods to produce a reassortant
influenza A virus which comprises at least one genome segment of a
cold-adapted equine influenza virus, where the equine influenza
genome segment confers on the reassortant virus at least one
identifying phenotype of the cold-adapted equine influenza
virus.
[0008] A cold-adapted equine influenza virus is one that replicates
in embryonated chicken eggs at a temperature ranging from about
26.degree. C. to about 30.degree. C. Preferably, a cold-adapted
equine influenza virus, reassortant influenza A virus, or
genetically-engineered equine influenza virus of the present
invention is attenuated, such that it will not cause disease in a
healthy animal.
[0009] In one embodiment, a cold-adapted equine influenza virus,
reassortant influenza A virus, or genetically-engineered equine
influenza virus of the present invention is also temperature
sensitive, such that the virus replicates in embryonated chicken
eggs at a temperature ranging from about 26.degree. C. to about
30.degree. C., forms plaques in tissue culture cells at a
permissive temperature of about 34.degree. C., but does not form
plaques in tissue culture cells at a non-permissive temperature of
about 39.degree. C.
[0010] In one embodiment, such a temperature sensitive virus
comprises two mutations: a first mutation that inhibits plaque
formation at a temperature of about 39.degree. C., that mutation
co-segregating with the genome segment that encodes the viral
nucleoprotein gene; and a second mutation that inhibits all viral
protein synthesis at a temperature of about 39.degree. C.
[0011] In another embodiment, a cold-adapted, temperature sensitive
equine influenza virus of the present invention replicates in
embryonated chicken eggs at a temperature ranging from about
26.degree. C. to about 30.degree. C., forms plaques in tissue
culture cells at a permissive temperature of about 34.degree. C.,
but does not form plaques in tissue culture cells or express late
viral proteins at a non-permissive temperature of about 37.degree.
C.
[0012] Typically, a cold-adapted equine influenza virus of the
present invention is produced by passaging a wild-type equine
influenza virus one or more times, and then selecting viruses that
stably grow and replicate at a reduced temperature. A cold-adapted
equine influenza virus produced thereby includes, in certain
embodiments, a dominant interference phenotype, that is, the virus,
when co-infected with a parental equine influenza virus or
heterologous wild-type influenza A virus, will inhibit the growth
of that virus.
[0013] Examples of cold-adapted equine influenza viruses of the
present invention include EIV-P821, identified by accession No.
ATCC VR 2625; EIV-P824, identified by accession No. ATCC VR 2624;
EIV-MSV+5, identified by accession No. ATCC VR 2627; and progeny of
such viruses.
[0014] Therapeutic compositions of the present invention include
from about 10.sup.5 TCID.sub.50 units to about 10.sup.8 TCID.sub.50
units, and preferably about 2.times.10.sup.6 TCID.sub.50 units, of
a cold-adapted equine influenza virus, reassortant influenza A
virus, or genetically-engineered equine influenza virus of the
present invention.
[0015] The present invention also includes a method to protect an
animal from disease caused by an influenza A virus, which includes
the step of administering to the animal a therapeutic composition
including a cold-adapted equine influenza virus, a reassortant
influenza A virus, or a genetically-engineered equine influenza
virus of the present invention.
[0016] Also provided is a method to treat animals for respiratory
disease which includes the administration of a therapeutic
composition of the present invention. While not being bound by
theory, it is believed that administration of a therapeutic
composition of the present invention exhibits non-specific
interference of respiratory disease in animals infected with equine
influenza virus or other infectious agents. Examples of such
infectious agents include, but are not limited to, any virus or
bacterium that leads to respiratory disease, either directly or
indirectly. The present invention provides a method to treat
animals that exhibit respiratory disease by administration of a
therapeutic composition including a cold-adapted equine influenza
virus, a reassortant influenza A virus, or a genetically-engineered
equine influenza virus of the present invention. The present
invention further provides a method to reduce nasal discharge in
young animals, such as that apparently caused by viral and/or
bacterial infections, by administration of a therapeutic
composition including a cold-adapted equine influenza virus, a
reassortant influenza A virus, or a genetically-engineered equine
influenza virus of the present invention. Preferred animals to
protect include equids, with horses and ponies being particularly
preferred.
[0017] Yet another embodiment of the present invention is a method
to generate a cold-adapted equine influenza virus. The method
includes the steps of passaging a wild-type equine influenza virus;
and selecting viruses that grow at a reduced temperature. In one
embodiment, the method includes repeating the passaging and
selection steps one or more times, while progressively reducing the
temperature. Passaging of equine influenza virus preferably takes
place in embryonated chicken eggs.
[0018] Another embodiment is an method to produce a reassortant
influenza A virus through genetic reassortment of the genome
segments of a donor cold-adapted equine influenza virus of the
present invention with the genome segments of a recipient influenza
A virus. Reassortant influenza A viruses of the present invention
are produced by a method that includes the steps of: (a) mixing the
genome segments of a donor cold-adapted equine influenza virus with
the genome segments of a recipient influenza A virus, and (b)
selecting viruses which include at least one identifying phenotype
of the donor equine influenza virus. Identifying phenotypes include
cold-adaptation, temperature sensitivity, dominant interference,
and attenuation. Preferably, such reassortant viruses at least
include the attenuation phenotype of the donor virus. A typical
reassortant virus will have the antigenicity of the recipient
virus, that is, it will retain the hemagglutinin (HA) and
neuraminidase (NA) phenotypes of the recipient virus.
[0019] The present invention provides methods to propagate
cold-adapted equine influenza viruses or reassortant influenza A
viruses of the present invention. These methods include propagation
in embryonated chicken eggs or in tissue culture cells.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The present invention provides experimentally-generated
cold-adapted equine influenza viruses comprising certain defined
phenotypes, which are disclosed herein. It is to be noted that the
term "a" or "an" entity, refers to one or more of that entity; for
example, "a cold-adapted equine influenza virus" can include one or
more cold-adapted equine influenza viruses. As such, the terms "a"
(or "an"), "one or more," and "at least one" can be used
interchangeably herein. It is also to be noted that the terms
"comprising," "including," and "having" can be used
interchangeably. Furthermore, an item "selected from the group
consisting of" refers to one or more of the items in that group,
including combinations thereof.
[0021] A cold-adapted equine influenza virus of the present
invention is a virus that has been generated in the laboratory, and
as such, is not a virus as occurs in nature. Since the present
invention also includes those viruses having the identifying
phenotypes of such a cold-adapted equine influenza virus, an equine
influenza virus isolated from a mixture of naturally-occurring
viruses, i.e., removed from its natural milieu, but having the
claimed phenotypes, is included in the present invention. A
cold-adapted equine influenza virus of the present invention does
not require any specific level of purity. For example, a
cold-adapted equine influenza virus grown in embryonated chicken
eggs may be in a mixture with the allantoic fluid (AF), and a
cold-adapted equine influenza virus grown in tissue culture cells
may be in a mixture with disrupted cells and tissue culture
medium.
[0022] As used herein, an "equine influenza virus" is an influenza
virus that infects and grows in equids, e.g., horses or ponies. As
used herein, "growth" of a virus denotes the ability of the virus
to reproduce or "replicate" itself in a permissive host cell. As
such, the terms, "growth of a virus" and "replication of a virus"
are used interchangeably herein. Growth or replication of a virus
in a particular host cell can be demonstrated and measured by
standard methods well-known to those skilled in the art of
virology. For example, samples containing infectious virus, e.g.,
as contained in nasopharyngeal secretions from an infected horse,
are tested for their ability to cause cytopathic effect (CPE),
e.g., virus plaques, in tissue culture cells. Infectious virus may
also be detected by inoculation of a sample into the allantoic
cavity of embryonated chicken eggs, and then testing the AF of eggs
thus inoculated for its ability to agglutinate red blood cells,
i.e., cause hemagglutination, due to the presence of the influenza
virus hemagglutinin (HA) protein in the AF.
[0023] Naturally-occurring, i.e., wild-type, equine influenza
viruses replicate well at a temperature from about 34.degree. C. to
about 39.degree. C. For example, wild-type equine influenza virus
replicates in embryonated chicken eggs at a temperature of about
34.degree. C., and replicates in tissue culture cells at a
temperature from about 34.degree. C. to about 39.degree. C. As used
herein, a "cold-adapted" equine influenza virus is an equine
influenza virus that has been adapted to grow at a temperature
lower than the optimal growth temperature for equine influenza
virus. One example of a cold-adapted equine influenza virus of the
present invention is a virus that replicates in embryonated chicken
eggs at a temperature of about 30.degree. C. A preferred
cold-adapted equine influenza virus of the present invention
replicates in embryonated chicken eggs at a temperature of about
28.degree. C. Another preferred cold-adapted equine influenza virus
of the present invention replicates in embryonated chicken eggs at
a temperature of about 26.degree. C. In general, preferred
cold-adapted equine influenza viruses of the present invention
replicate in embryonated chicken eggs at a temperature ranging from
about 26.degree. C. to about 30.degree. C., i.e., at a range of
temperatures at which a wild-type virus will grow poorly or not at
all. It should be noted that the ability of such viruses to
replicate within that temperature range does not preclude their
ability to also replicate at higher or lower temperatures. For
example, one embodiment is a cold-adapted equine influenza virus
that replicates in embryonated chicken eggs at a temperature of
about 26.degree. C., but also replicates in tissue culture cells at
a temperature of about 34.degree. C. As with wild-type equine
influenza viruses, cold-adapted equine influenza viruses of the
present invention generally form plaques in tissue culture cells,
for example Madin Darby Canine Kidney Cells (MDCK) at a temperature
of about 34.degree. C. Examples of suitable and preferred
cold-adapted equine influenza viruses of the present invention are
disclosed herein.
[0024] One embodiment of the present invention is a cold-adapted
equine influenza virus that is produced by a method which includes
passaging a wild-type equine influenza virus, and then selecting
viruses that grow at a reduced temperature. Cold-adapted equine
influenza viruses of the present invention can be produced, for
example, by sequentially passaging a wild-type equine influenza
virus in embryonated chicken eggs at progressively lower
temperatures, thereby selecting for certain members of the virus
mixture which stably replicate at the reduced temperature. An
example of a passaging procedure is disclosed in detail in the
Examples section. During the passaging procedure, one or more
mutations appear in certain of the single-stranded RNA segments
comprising the influenza virus genome, which alter the genotype,
i.e., the primary nucleotide sequence of those RNA segments. As
used herein, a "mutation" is an alteration of the primary
nucleotide sequence of any given RNA segment making up an influenza
virus genome. Examples of mutations include substitution of one or
more nucleotides, deletion of one or more nucleotides, insertion of
one or more nucleotides, or inversion of a stretch of two or more
nucleotides. By selecting for those members of the virus mixture
that stably replicate at a reduced temperature, a virus with a
cold-adaptation phenotype is selected. As used herein, a
"phenotype" is an observable or measurable characteristic of a
biological entity such as a cell or a virus, where the observed
characteristic is attributable to a specific genetic configuration
of that biological entity, i.e., a certain genotype. As such, a
cold-adaptation phenotype is the result of one or more mutations in
the virus genome. As used herein, the terms "a mutation," "a
genome," "a genotype," or "a phenotype" refer to one or more, or at
least one mutation, genome, genotype, or phenotype,
respectively.
[0025] Additional, observable phenotypes in a cold-adapted equine
influenza virus may occur, and will generally be the result of one
or more additional mutations in the genome of such a virus. For
example, a cold-adapted equine influenza virus of the present
invention may, in addition, be attenuated, exhibit dominant
interference, and/or be temperature sensitive.
[0026] In one embodiment, a cold-adapted equine influenza virus of
the present invention has a phenotype characterized by attenuation.
A cold-adapted equine influenza virus is "attenuated," when
administration of the virus to an equine influenza
virus-susceptible animal results in reduced or absent clinical
signs in that animal, compared to clinical signs observed in
animals that are infected with wild-type equine influenza virus.
For example, an animal infected with wild-type equine influenza
virus will display fever, sneezing, coughing, depression, and nasal
discharges. In contrast, an animal administered an attenuated,
cold-adapted equine influenza virus of the present invention will
display minimal or no, i.e., undetectable, clinical disease
signs.
[0027] In another embodiment, a cold-adapted equine influenza virus
of the present invention comprises a temperature sensitive
phenotype. As used herein, a temperature sensitive cold-adapted
equine influenza virus replicates at reduced temperatures, but no
longer replicates or forms plaques in tissue culture cells at
certain higher growth temperatures at which the wild-type virus
will replicate and form plaques. While not being bound by theory,
it is believed that replication of equine influenza viruses with a
temperature sensitive phenotype is largely restricted to the cool
passages of the upper respiratory tract, and does not replicate
efficiently in the lower respiratory tract, where the virus is more
prone to cause disease symptoms. A temperature at which a
temperature sensitive virus will grow is referred to herein as a
"permissive" temperature for that temperature sensitive virus, and
a higher temperature at which the temperature sensitive virus will
not grow, but at which a corresponding wild-type virus will grow,
is referred to herein as a "non-permissive" temperature for that
temperature sensitive virus. For example, certain temperature
sensitive cold-adapted equine influenza viruses of the present
invention replicate in embryonated chicken eggs at a temperature at
or below about 30.degree. C., preferably at about 28.degree. C. or
about 26.degree. C., and will form plaques in tissue culture cells
at a permissive temperature of about 34.degree. C., but will not
form plaques in tissue culture cells at a non-permissive
temperature of about 39.degree. C. Other temperature sensitive
cold-adapted equine influenza viruses of the present invention
replicate in embryonated chicken eggs at a temperature at or below
about 30.degree. C., preferably at about 28.degree. C. or about
26.degree. C., and will form plaques in tissue culture cells at a
permissive temperature of about 34.degree. C., but will not form
plaques in tissue culture cells at a non-permissive temperature of
about 37.degree. C.
[0028] Certain cold-adapted equine influenza viruses of the present
invention have a dominant interference phenotype; that is, they
dominate an infection when co-infected into cells with another
influenza A virus, thereby impairing the growth of that other
virus. For example, when a cold-adapted equine influenza virus of
the present invention, having a dominant interference phenotype, is
co-infected into MDCK cells with the wild-type parental equine
influenza virus, A/equine/Kentucky/1/91 (H3N8), growth of the
parental virus is impaired. Thus, in an animal that has recently
been exposed to, or may be soon exposed to, a virulent influenza
virus, i.e., an influenza virus that causes disease symptoms,
administration of a therapeutic composition comprising a
cold-adapted equine influenza virus having a dominant interference
phenotype into the upper respiratory tract of that animal will
impair the growth of the virulent virus, thereby ameliorating or
reducing disease in that animal, even in the absence of an immune
response to the virulent virus.
[0029] Dominant interference of a cold-adapted equine influenza
virus having a temperature sensitive phenotype can be measured by
standard virological methods. For example, separate monolayers of
MDCK cells can be infected with (a) a virulent wild-type influenza
A virus, (b) a temperature sensitive, cold-adapted equine influenza
virus, and (c) both viruses in a co-infection, with all infections
done at multiplicities of infection (MOI) of about 2 plaque forming
units (pfu) per cell. After infection, the virus yields from the
various infected cells are measured by duplicate plaque assays
performed at the permissive temperature for the cold-adapted equine
influenza virus and at the non-permissive temperature of that
virus. A cold adapted equine influenza virus having a temperature
sensitive phenotype is unable to form plaques at its non-permissive
temperature, while the wild-type virus is able to form plaques at
both the permissive and non-permissive temperatures. Thus it is
possible to measure the growth of the wild-type virus in the
presence of the cold adapted virus by comparing the virus yield at
the non-permissive temperature of the cells singly infected with
wild-type virus to the yield at the non-permissive temperature of
the wild-type virus in doubly infected cells.
[0030] Cold-adapted equine influenza viruses of the present
invention are characterized primarily by one or more of the
following identifying phenotypes: cold-adaptation, temperature
sensitivity, dominant interference, and/or attenuation. As used
herein, the phrase "an equine influenza virus comprises the
identifying phenotype(s) of cold-adaptation, temperature
sensitivity, dominant interference, and/or attenuation" refers to a
virus having such a phenotype(s). Examples of such viruses include,
but are not limited to, EIV-P821, identified by accession No. ATCC
VR 2625, EIV-P824, identified by accession No. ATCC VR 2624, and
EIV-MSV+5, identified by accession No. ATCC VR 2627, as well as
EIV-MSV0, EIV, MSV+1, EIV-MSV+2, EIV-MSV+3, and EIV-MSV+4.
Production of such viruses is described in the examples. For
example, cold-adapted equine influenza virus EIV-P821 is
characterized by, i.e., has the identifying phenotypes of, (a)
cold-adaptation, e.g., its ability to replicate in embryonated
chicken eggs at a temperature of about 26.degree. C.; (b)
temperature sensitivity, e.g., its inability to form plaques in
tissue culture cells and to express late gene products at a
non-permissive temperature of about 37.degree. C., and its
inability to form plaques in tissue culture cells and to synthesize
any viral proteins at a non-permissive temperature of about
39.degree. C.; (c) its attenuation upon administration to an equine
influenza virus-susceptible animal; and (d) dominant interference,
e.g., its ability, when co-infected into a cell with a wild-type
influenza A virus, to interfere with the growth of that wild-type
virus. Similarly, cold-adapted equine influenza virus EIV-P824 is
characterized by (a) cold adaptation, e.g., its ability to
replicate in embryonated chicken eggs at a temperature of about
28.degree. C.; (b) temperature sensitivity, e.g., its inability to
form plaques in tissue culture cells at a non-permissive
temperature of about 39.degree. C.; and (c) dominant interference,
e.g., its ability, when co-infected into a cell with a wild-type
influenza A virus, to interfere with the growth of that wild-type
virus. In another example, cold-adapted equine influenza virus
EIV-MSV+5 is characterized by (a) cold-adaptation, e.g., its
ability to replicate in embryonated chicken eggs at a temperature
of about 26.degree. C.; (b) temperature sensitivity, e.g., its
inability to form plaques in tissue culture cells at a
non-permissive temperature of about 39.degree. C.; and (c) its
attenuation upon administration to an equine influenza
virus-susceptible animal.
[0031] In certain cases, the RNA segment upon which one or more
mutations associated with a certain phenotype occur may be
determined through reassortment analysis by standard methods, as
disclosed herein. In one embodiment, a cold-adapted equine
influenza virus of the present invention comprises a temperature
sensitive phenotype that correlates with at least two mutations in
the genome of that virus. In this embodiment, one of the two
mutations, localized by reassortment analysis as disclosed herein,
inhibits, i.e., blocks or prevents, the ability of the virus to
form plaques in tissue culture cells at a non-permissive
temperature of about 39.degree. C. This mutation co-segregates with
the segment of the equine influenza virus genome that encodes the
nucleoprotein (NP) gene of the virus, i.e., the mutation is located
on the same RNA segment as the NP gene. In this embodiment, the
second mutation inhibits all protein synthesis at a non-permissive
temperature of about 39.degree. C. As such, at the non-permissive
temperature, the virus genome is incapable of expressing any viral
proteins. Examples of cold-adapted equine influenza viruses
possessing these characteristics are EIV-P821 and EIV MSV+5.
EIV-P821 was generated by serial passaging of a wild-type equine
influenza virus in embryonated chicken eggs by methods described in
Example 1A. EIV-MSV+5 was derived by further serial passaging of
EIV-P821, as described in Example 1E.
[0032] Furthermore, a cold-adapted, temperature sensitive equine
influenza virus comprising the two mutations which inhibit plaque
formation and viral protein synthesis at a non-permissive
temperature of about 39.degree. C. can comprise one or more
additional mutations, which inhibit the virus' ability to
synthesize late gene products and to form plaques in tissue culture
cells at a non-permissive temperature of about 37.degree. C. An
example of a cold-adapted equine influenza virus possessing these
characteristics is EIV-P821. This virus isolate replicates in
embryonated chicken eggs at a temperature of about 26.degree. C.,
and does not form plaques or express any viral proteins at a
temperature of about 39.degree. C. Furthermore, EIV-P821 does not
form plaques on MDCK cells at a non-permissive temperature of about
37.degree. C., and at this temperature, late gene expression is
inhibited in such a way that late proteins are not produced, i.e.,
normal levels of NP protein are synthesized, reduced or
undetectable levels of M1 or HA proteins are synthesized, and
enhanced levels of the polymerase proteins are synthesized. Since
this phenotype is typified by differential viral protein synthesis,
it is distinct from the protein synthesis phenotype seen at a
non-permissive temperature of about 39.degree. C., which is
typified by the inhibition of synthesis of all viral proteins.
[0033] Pursuant to 37 CFR .sctn. 1.802 (a-c), cold-adapted equine
influenza viruses, designated herein as EIV-P821, an EIV-P824 were
deposited with the American Type Culture Collection (ATCC, 10801
University Boulevard, Manassas, Va. 20110-2209) under the Budapest
Treaty as ATCC Accession Nos. ATCC VR-2625, and ATCC VR-2624,
respectively, on Jul. 11, 1998. Cold-adapted equine influenza virus
EIV-MSV+5 was deposited with the ATCC as ATCC Accession No. ATCC
VR-2627 on Aug. 3, 1998. Pursuant to 37 CFR .sctn. 1.806, the
deposits are made for a term of at least thirty (30) years and at
least five (5) years after the most recent request for the
furnishing of a sample of the deposit was received by the
depository. Pursuant to 37 CFR .sctn. 1.808 (a)(2), all
restrictions imposed by the depositor on the availability to the
public will be irrevocably removed upon the granting of the
patent.
[0034] Preferred cold-adapted equine influenza viruses of the
present invention have the identifying phenotypes of EIV-P821,
EIV-P824, and EIV-MSV+5. Particularly preferred cold-adapted equine
influenza viruses include EIV-P821, EIV-P824, EIV-MSV+5, and
progeny of these viruses. As used herein, "progeny" are
"offspring," and as such can slightly altered phenotypes compared
to the parent virus, but retain identifying phenotypes of the
parent virus, for example, cold-adaptation, temperature
sensitivity, dominant interference, or attenuation. For example,
cold-adapted equine influenza virus EIV-MSV+5 is a "progeny" of
cold-adapted equine influenza virus EIV-P821. "Progeny" also
include reassortant influenza A viruses that comprise one or more
identifying phenotypes of the donor parent virus.
[0035] Reassortant influenza A viruses of the present invention are
produced by genetic reassortment of the genome segments of a donor
cold-adapted equine influenza virus of the present invention with
the genome segments of a recipient influenza A virus, and then
selecting a reassortant virus that derives at least one of its
eight RNA genome segments from the donor virus, such that the
reassortant virus acquires at least one identifying phenotype of
the donor cold-adapted equine influenza virus. Identifying
phenotypes include cold-adaptation, temperature sensitivity,
attenuation, and dominant interference. Preferably, reassortant
influenza A viruses of the present invention derive at least the
attenuation phenotype of the donor virus. Methods to isolate
reassortant influenza viruses are well known to those skilled in
the art of virology and are disclosed, for example, in Fields, et
al., 1996, Fields Virology, 3d ed., Lippincott-Raven; and Palese,
et al., 1976, J. Virol., 17, 876-884. Fields, et al., ibid. and
Palese, et al., ibid. are incorporated herein by reference in their
entireties.
[0036] A suitable donor equine influenza virus is a cold-adapted
equine influenza virus of the present invention, for example,
EIV-P821, identified by accession No. ATCC VR2625, EIV-P824,
identified by accession No. ATCC VR2624, or EIV-MSV+5, identified
by accession No. ATCC VR2627. A suitable recipient influenza A
virus can be another equine influenza virus, for example a Eurasian
subtype 2 equine influenza virus such as A/equine/Suffolk/89 (H3N8)
or a subtype 1 equine influenza virus such as A/Prague/1/56 (H7N7).
A recipient influenza A virus can also be any influenza A virus
capable of forming a reassortant virus with a donor cold-adapted
equine influenza virus. Examples of such influenza A viruses
include, but are not limited to, human influenza viruses such as
A/Puerto Rico/8/34 (H1N1), A/Hong Kong/156/97 (H5N1),
A/Singapore/1/57 (H2N2), and A/Hong Kong/1/68 (H3N2); swine viruses
such as A/Swine/Iowa/15/30 (H1N1); and avian viruses such as
A/mallard/New York/6750/78 (H2N2) and A/chicken/Hong Kong/258/97
(H5N1). A reassortant virus of the present invention can include
any combination of donor and recipient gene segments, as long as
the resulting reassortant virus possesses at least one identifying
phenotype of the donor virus.
[0037] One example of a reassortant virus of the present invention
is a "6+2" reassortant virus, in which the six "internal gene
segments," i.e., those comprising the NP, PB2, PB 1, PA, M, and NS
genes, are derived from the donor cold-adapted equine influenza
virus genome, and the two "external gene segments," i.e., those
comprising the HA and NA genes, are derived from the recipient
influenza A virus. A resultant virus thus produced has the
attenuated, cold-adapted, temperature sensitive, and/or dominant
interference phenotypes of the donor cold-adapted equine influenza
virus, but the antigenicity of the recipient strain.
[0038] In yet another embodiment, a cold-adapted equine influenza
virus of the present invention can be produced through recombinant
means. In this approach, one or more specific mutations, associated
with identified cold-adaptation, attenuation, temperature
sensitivity, or dominant interference phenotypes, are identified
and are introduced back into a wild-type equine influenza virus
strain using a reverse genetics approach. Reverse genetics entails
using RNA polymerase complexes isolated from influenza
virus-infected cells to transcribe artificial influenza virus
genome segments containing the mutation(s), incorporating the
synthesized RNA segment(s) into virus particles using a helper
virus, and then selecting for viruses containing the desired
changes. Reverse genetics methods for influenza viruses are
described, for example, in Enami, et al., 1990, Proc. Natl. Acad.
Sci. 87, 3802-3805; and in U.S. Pat. No. 5,578,473, by Palese, et
al., issued Nov. 26, 1996, both of which are incorporated herein by
reference in their entireties. This approach allows one skilled in
the art to produce additional cold-adapted equine influenza viruses
of the present invention without the need to go through the lengthy
cold-adaptation process, and the process of selecting mutants both
in vitro and in vivo with the desired virus phenotype.
[0039] A cold-adapted equine influenza virus of the present
invention may be propagated by standard virological methods
well-known to those skilled in the art, examples of which are
disclosed herein. For example, a cold-adapted equine influenza
virus can be grown in embryonated chicken eggs or in eukaryotic
tissue culture cells. Suitable continuous eukaryotic cell lines
upon which to grow a cold-adapted equine influenza virus of the
present invention include those that support growth of influenza
viruses, for example, MDCK cells. Other suitable cells upon which
to grow a cold-adapted equine influenza virus of the present
invention include, but are not limited to, primary kidney cell
cultures of monkey, calf, hamster or chicken.
[0040] In one embodiment, the present invention provides a
therapeutic composition to protect an animal against disease caused
by an influenza A virus, where the therapeutic composition includes
either a cold-adapted equine influenza virus or a reassortant
influenza A virus comprising at least one genome segment of an
equine influenza virus generated by cold-adaptation, wherein the
equine influenza virus genome segment confers at least one
identifying phenotype of the cold-adapted equine influenza virus.
In addition, a therapeutic composition of the present invention can
include an equine influenza virus that has been genetically
engineered to comprise one or more mutations, where those mutations
have been identified to confer a certain identifying phenotype on a
cold-adapted equine influenza virus of the present invention. As
used herein, the phrase "disease caused by an influenza A virus"
refers to the clinical manifestations observed in an animal which
has been infected with a virulent influenza A virus. Examples of
such clinical manifestations include, but are not limited to,
fever, sneezing, coughing, nasal discharge, rales, anorexia and
depression. In addition, the phrase "disease caused by an influenza
A virus" is defined herein to include shedding of virulent virus by
the infected animal. Verification that clinical manifestations
observed in an animal correlate with infection by virulent equine
influenza virus may be made by several methods, including the
detection of a specific antibody and/or T-cell responses to equine
influenza virus in the animal. Preferably, verification that
clinical manifestations observed in an animal correlate with
infection by a virulent influenza A virus is made by the isolation
of the virus from the afflicted animal, for example, by swabbing
the nasopharyngeal cavity of that animal for virus-containing
secretions. Verification of virus isolation may be made by the
detection of CPE in tissue culture cells inoculated with the
isolated secretions, by inoculation of the isolated secretions into
embryonated chicken eggs, where virus replication is detected by
the ability of AF from the inoculated eggs to agglutinate
erythrocytes, suggesting the presence of the influenza virus
hemagglutinin protein, or by use of a commercially available
diagnostic test, for example, the Directigen.RTM. FLU A test.
[0041] As used herein, the term "to protect" includes, for example,
to prevent or to treat influenza A virus infection in the subject
animal. As such, a therapeutic composition of the present invention
can be used, for example, as a prophylactic vaccine to protect a
subject animal from influenza disease by administering the
therapeutic composition to that animal at some time prior to that
animal's exposure to the virulent virus.
[0042] A therapeutic composition of the present invention,
comprising a cold-adapted equine influenza virus having a dominant
interference phenotype, can also be used to treat an animal that
has been recently infected with virulent influenza A virus or is
likely to be subsequently exposed in a few days, such that the
therapeutic composition immediately interferes with the growth of
the virulent virus, prior to the animal's production of antibodies
to the virulent virus. A therapeutic composition comprising a
cold-adapted equine influenza virus having a dominant interference
phenotype may be effectively administered prior to subsequent
exposure for a length of time corresponding to the approximate
length of time that a cold-adapted equine influenza virus of the
present invention will replicate in the upper respiratory tract of
a treated animal, for example, up to about seven days. A
therapeutic composition comprising a cold-adapted equine influenza
virus having a dominant interference phenotype may be effectively
administered following exposure to virulent equine influenza virus
for a length of time corresponding to the time required for an
infected animal to show disease symptoms, for example, up to about
two days.
[0043] Therapeutic compositions of the present invention can be
administered to any animal susceptible to influenza virus disease,
for example, humans, swine, horses and other equids, aquatic birds,
domestic and game fowl, seals, mink, and whales. Preferably, a
therapeutic composition of the present invention is administered
equids. Even more preferably, a therapeutic composition of the
present invention is administered to a horse, to protect against
equine influenza disease.
[0044] Current vaccines available to protect horses against equine
influenza virus disease are not effective in protecting young
foals, most likely because they cannot overcome the maternal
antibody present in these young animals, and often, vaccination at
an early age, for example 3 months of age, can lead to tolerance
rather than immunity. In one embodiment, and in contrast to
existing equine influenza virus vaccines, a therapeutic composition
comprising a cold-adapted equine influenza virus of the present
invention apparently can produce immunity in young animals. As
such, a therapeutic composition of the present invention can be
safely and effectively administered to young foals, as young as
about 3 months of age, to protect against equine influenza disease
without the induction of tolerance.
[0045] In one embodiment, a therapeutic composition of the present
invention can be multivalent. For example, it can protect an animal
from more than one strain of influenza A virus by providing a
combination of one or more cold-adapted equine influenza viruses of
the present invention, one or more reassortant influenza A viruses,
and/or one or more genetically-engineered equine influenza viruses
of the present invention. Multivalent therapeutic compositions can
include at least two cold-adapted equine influenza viruses, e.g.,
against North American subtype-2 virus isolates such as
A/equine/Kentucky/1/91 (H1N8), and Eurasian subtype-2 virus
isolates such as A/equine/Suffolk/89 (H3N8); or one or more
subtype-2 virus isolates and a subtype-I virus isolate such as
A/equine/Prague/1/56 (H7N7). Similarly, a multivalent therapeutic
composition of the present invention can include a cold-adapted
equine influenza virus and a reassortant influenza A virus of the
present invention, or two reassortant influenza A viruses of the
present invention. A multivalent therapeutic composition of the
present invention can also contain one or more formulations to
protect against one or more other infectious agents in addition to
influenza A virus. Such other infectious agents include, but not
limited to: viruses; bacteria; fungi and fungal-related
microorganisms; and parasites. Preferable multivalent therapeutic
compositions include, but are not limited to, a cold-adapted equine
influenza virus, reassortant influenza A virus, or
genetically-engineered equine influenza virus of the present
invention plus one or more compositions protective against one or
more other infectious agents that afflict horses. Suitable
infectious agents to protect against include, but are not limited
to, equine infectious anemia virus, equine herpes virus, eastern,
western, or Venezuelan equine encephalitis virus, tetanus,
Streptococcus equi, and Ehrlichia resticii.
[0046] A therapeutic composition of the present invention can be
formulated in an excipient that the animal to be treated can
tolerate. Examples of such excipients include water, saline,
Ringer's solution, dextrose solution, Hank's solution, and other
aqueous physiologically balanced salt solutions. Excipients can
also contain minor amounts of additives, such as substances that
enhance isotonicity and chemical or biological stability. Examples
of buffers include phosphate buffer, bicarbonate buffer, and Tris
buffer, while examples of stabilizers include A1/A2 stabilizer,
available from Diamond Animal Health, Des Moines, Iowa. Standard
formulations can either be liquids or solids which can be taken up
in a suitable liquid as a suspension or solution for administration
to an animal. In one embodiment, a non-liquid formulation may
comprise the excipient salts, buffers, stabilizers, etc., to which
sterile water or saline can be added prior to administration.
[0047] A therapeutic composition of the present invention may also
include one or more adjuvants or carriers. Adjuvants are typically
substances that enhance the immune response of an animal to a
specific antigen, and carriers include those compounds that
increase the half-life of a therapeutic composition in the treated
animal. One advantage of a therapeutic composition comprising a
cold-adapted equine influenza virus or a reassortant influenza A
virus of the present invention is that adjuvants and carriers are
not required to produce an efficacious vaccine. Furthermore, in
many cases known to those skilled in the art, the advantages of a
therapeutic composition of the present invention would be hindered
by the use of some adjuvants or carriers. However, it should be
noted that use of adjuvants or carriers is not precluded by the
present invention.
[0048] Therapeutic compositions of the present invention include an
amount of a cold-adapted equine influenza virus that is sufficient
to protect an animal from challenge with virulent equine influenza
virus. In one embodiment, a therapeutic composition of the present
invention can include an amount of a cold-adapted equine influenza
virus ranging from about 10.sup.5 tissue culture infectious dose-50
(TCID.sub.50) units of virus to about 10.sup.8 TCID.sub.50 units of
virus. As used herein, a "TCID.sub.50 unit" is amount of a virus
which results in cytopathic effect in 50% of those cell cultures
infected. Methods to measure and calculate TCID.sub.50 are known to
those skilled in the art and are available, for example, in Reed
and Muench, 1938, Am. J. of Hyg. 27, 493-497, which is incorporated
herein by reference in its entirety. A preferred therapeutic
composition of the present invention comprises from about 10.sup.6
TCID.sub.50 units to about 10.sup.7 TCID.sub.50 units of a
cold-adapted equine influenza virus or reassortant influenza A
virus of the present invention. Even more preferred is a
therapeutic composition comprising about 2.times.10.sup.6
TCID.sub.50 units of a cold-adapted equine influenza virus or
reassortant influenza A virus of the present invention.
[0049] The present invention also includes methods to protect an
animal against disease caused by an influenza A virus comprising
administering to the animal a therapeutic composition of the
present invention. Preferred are those methods which protect an
equid against disease caused by equine influenza virus, where those
methods comprise administering to the equid a cold-adapted equine
influenza virus. Acceptable protocols to administer therapeutic
compositions in an effective manner include individual dose size,
number of doses, frequency of dose administration, and mode of
administration. Determination of such protocols can be accomplished
by those skilled in the art, and examples are disclosed herein.
[0050] A preferable method to protect an animal against disease
caused by an influenza A virus includes administering to that
animal a single dose of a therapeutic composition comprising a
cold-adapted equine influenza virus, a reassortant influenza A
virus, or genetically-engineered equine influenza virus of the
present invention. A suitable single dose is a dose that is capable
of protecting an animal from disease when administered one or more
times over a suitable time period. The method of the present
invention may also include administering subsequent, or booster
doses of a therapeutic composition. Booster administrations can be
given from about 2 weeks to several years after the original
administration. Booster administrations preferably are administered
when the immune response of the animal becomes insufficient to
protect the animal from disease. Examples of suitable and preferred
dosage schedules are disclosed in the Examples section.
[0051] A therapeutic composition of the present invention can be
administered to an animal by a variety of means, such that the
virus will enter and replicate in the mucosal cells in the upper
respiratory tract of the treated animal. Such means include, but
are not limited to, intranasal administration, oral administration,
and intraocular administration. Since influenza viruses naturally
infect the mucosa of the upper respiratory tract, a preferred
method to administer a therapeutic composition of the present
invention is by intranasal administration. Such administration may
be accomplished by use of a syringe fitted with cannula, or by use
of a nebulizer fitted over the nose and mouth of the animal to be
vaccinated.
[0052] The efficacy of a therapeutic composition of the present
invention to protect an animal against disease caused by influenza
A virus can be tested in a variety of ways including, but not
limited to, detection of antibodies by, for example,
hemagglutination inhibition (HAI) tests, detection of cellular
immunity within the treated animal, or challenge of the treated
animal with virulent equine influenza virus to determine whether
the treated animal is resistant to the development of disease. In
addition, efficacy of a therapeutic composition of the present
invention comprising a cold-adapted equine influenza virus having a
dominant interference phenotype to ameliorate or reduce disease
symptoms in an animal previously inoculated or susceptible to
inoculation with a virulent, wild-type equine influenza virus can
be tested by screening for the reduction or absence of disease
symptoms in the treated animal.
[0053] The present invention also includes methods to produce a
therapeutic composition of the present invention. Suitable and
preferred methods for making a therapeutic composition of the
present invention are disclosed herein. Pertinent steps involved in
producing one type of therapeutic composition of the present
invention, i.e., a cold-adapted equine influenza virus, include (a)
passaging a wild-type equine influenza virus in vitro, for example,
in embryonated chicken eggs; (b) selecting viruses that grow at a
reduced temperature; (c) repeating the passaging and selection
steps one or more times, at progressively lower temperatures, until
virus populations are selected which stably grow at the desired
lower temperature; and (d) mixing the resulting virus preparation
with suitable excipients.
[0054] The pertinent steps involved in producing another type of
therapeutic composition of the present invention, i.e., a
reassortant influenza A virus having at least one genome segment of
an equine influenza virus generated by adaptation, includes the
steps of (a) mixing the genome segments of a donor cold-adapted
equine influenza virus, which preferably also has the phenotypes of
attenuation, temperature sensitivity, or dominant interference,
with the genome segments of a recipient influenza A virus, and (b)
selecting reassortant viruses that have at least one identifying
phenotype of the donor equine influenza virus. Identifying
phenotypes to select for include attenuation, cold-adaptation,
temperature sensitivity, and dominant interference. Methods to
screen for these phenotypes are well known to those skilled in the
art, and are disclosed herein. It is preferable to screen for
viruses that at least have the phenotype of attenuation.
[0055] Using this method to generate a reassortant influenza A
virus having at least one genome segment of a equine influenza
virus generated by cold-adaptation, one type of reassortant virus
to select for is a "6+2" reassortant, where the six "internal gene
segments," i.e., those coding for the NP, PB2, PB 1, PA, M, and NS
genes, are derived from the donor cold-adapted equine influenza
virus genome, and the two "external gene segments," i.e., those
coding for the HA and NA genes, are derived from the recipient
influenza A virus. A resultant virus thus produced can have the
cold-adapted, attenuated, temperature sensitive, and/or
interference phenotypes of the donor cold-adapted equine influenza
virus, but the antigenicity of the recipient strain.
[0056] In another embodiment, a therapeutic composition comprising
a cold-adapted equine influenza virus of the present invention can
be administered to an animal to treat respiratory disease such as,
respiratory disease but not limited to, nasal discharge (also
referred to as "snots" by equine veterinarians) which is believed
to be due directly or indirectly to equine herpes virus (EHV) or
other viruses. As used herein, the term "treat" refers to the
ability of a therapeutic composition of the present invention to
reduce or eliminate disease, such as respiratory disease in an
animal. Such disease is preferably a pre-existing condition and/or
is preferably due to recent exposure or subsequent exposure within
a few days of treatment to an infectious agent able to cause such
disease. A therapeutic composition of the present invention can be
safely and effectively administered to equids, preferably equids
from about 3 months to about 2 years of age, to treat an infection
such as, but not limited to, any virus or bacterium that leads to
respiratory disease, preferably upper respiratory disease, either
directly or indirectly. Maternal immunity is believed to protect a
young animal through about the first three months of life, but
between about three to six months of age maternal immunity begins
to decrease leaving animals susceptible to infection. A therapeutic
composition of the present invention can effectively treat
respiratory disease in these animals. A therapeutic composition of
the present invention can also be used to inhibit secondary viral
and/or bacterial infection in animals. Furthermore a therapeutic
composition of the present invention may be used to treat other
diseases caused by infectious agents, either directly or
indirectly.
[0057] While not being bound by theory, it is believed that a
therapeutic composition of the present invention induces a
non-specific interference response which is not dependent on the
production of antibodies; rather this response is most likely due
to increased cytokine production triggered by the administration of
such a therapeutic composition of the present invention, leading to
amelioration of disease. Dominant interference refers herein to the
ability of an equine influenza virus of the present invention to
inhibit growth of the parental wild-type strain, as well as
heterologous influenza A viruses. As used herein, non-specific
interference refers to the ability of an equine influenza virus of
the present invention to inhibit growth of the parental wild-type
strain, as well as other infectious agents such as, but not limited
to viruses or bacteria, that lead to respiratory disease,
preferably before antibody-mediated immunity can be established in
response to administration of that virus.
[0058] A preferred embodiment comprises administering to an animal
a therapeutic composition of the present invention comprising a
virus selected from the group consisting of a cold-adapted equine
influenza virus having a dominant interference phenotype; and a
reassortant influenza A virus comprising a portion of an equine
influenza virus genome conferring cold-adaptation and dominant
interference phenotypes, wherein a therapeutic composition exhibits
non-specific interference. A portion refers herein to one or more
genome segments, or fragments; in this embodiment thereof that
encode the cold-adapted and dominant interference phenotypes.
[0059] Administration of a therapeutic composition of the present
invention to horses suffering from respiratory disease preferably
results in reduced signs of disease in less than about 7 days, more
preferably in less than 5 days and even more preferably within
about 1 to 3 days. The stage of disease can range from early onset
with symptoms of nasal discharge and fever to later stages with
nasal discharge as the predominant symptom.
[0060] A preferable method to treat an animal for disease caused by
respiratory disease includes administering to that animal a single
dose of a therapeutic composition comprising a cold-adapted equine
influenza virus, a reassortant influenza A virus, or
genetically-engineered equine influenza virus of the present
invention. Such administration can be accomplished as described
elsewhere herein. Examples include but are not limited to, use of a
syringe fitted with a cannula, or use of a nebulizer fitted over
the nose and mouth of the animal to be treated. A suitable dose is
a dose that is capable of treating an animal for disease when
administered one or more times over a suitable time period. A
preferred therapeutic composition of the present invention
comprises from about 10.sup.7TCID.sub.50 units to about 10.sup.8
TCID.sub.50 units of a cold-adapted equine influenza virus or
reassortant influenza A virus of the present invention. The method
of the present invention may also include administering subsequent
doses of a therapeutic composition.
[0061] In yet another embodiment, a virus of the present invention
can be used as an adjuvant to stimulate the immune response of a
vaccine against another infectious agent.
[0062] The following examples are provided for the purposes of
illustration and are not intended to limit the scope of the present
invention.
EXAMPLE 1
[0063] This example discloses the production and phenotypic
characterization of several cold-adapted equine influenza viruses
of the present invention.
[0064] A. Parental equine influenza virus, A/equine/Kentucky/1/91
(H3N8) (obtained from Tom Chambers, the University of Kentucky,
Lexington, Ky.) was subjected to cold-adaptation in a foreign host
species, i.e., embryonated chicken eggs, in the following manner.
Embryonated, 10 or 11-day old chicken eggs, available, for example,
from Truslow Farms, Chestertown, Md. or from HyVac, Adel, Iowa,
were inoculated with the parental equine influenza virus by
injecting about 0.1 milliliter (ml) undiluted AF containing
approximately 10.sup.6 plaque forming units (pfu) of virus into the
allantoic cavity through a small hole punched in the shell of the
egg. The holes in the eggs were sealed with nail polish and the
eggs were incubated in a humidified incubator set at the
appropriate temperature for three days. Following incubation, the
eggs were candled and any non-viable eggs were discarded. AF was
harvested from viable embryos by aseptically removing a portion of
the egg shell, pulling aside the chorioallantoic membrane (CAM)
with sterile forceps and removing the AF with a sterile pipette.
The harvested AF was frozen between passages. The AF was then used,
either undiluted or diluted 1:1000 in phosphate-buffered saline
(PBS) as noted in Table 1, to inoculate a new set of eggs for a
second passage, and so on. A total of 69 passages were completed.
Earlier passages were done at either about 34.degree. C. (passages
1-2) or about 30.degree. C. and on subsequent passages, the
incubation temperature was shifted down either to about 28.degree.
C., or to about 26.degree. C. In order to increase the possibility
of the selection of the desired phenotype of a stable, attenuated
virus, the initial serial passage was expanded to included five
different limbs of the serial passage tree, A through E, as shown
in Table 1.
1TABLE 1 Passage history of the limbs A through E Passage #
Temperature Limb A Limb B Limb C Limb D Limb E 34.degree. C. 1-2
1-2 1-2 1-2 1-2 30.degree. C. 3-8 3-29 3-29 3-29 3-29 28.degree. C.
30-33* 30-68* 30-33 30-69 26.degree. C. 9-65 34-69* 34-65 *=the
infectious allantoic fluid was diluted 1:1000 in these passages
[0065] B. Virus isolates carried through the cold-adaptation
procedure described in section A were tested for temperature
sensitivity, i.e., a phenotype in which the cold-adapted virus
grows at the lower, or permissive temperature (e.g., about
34.degree. C.), but no longer forms plaques at a higher, or
non-permissive temperature (e.g., about 37.degree. C. or about
39.degree. C.), as follows. At each cold-adaptation passage, the AF
was titered by plaque assay at about 34.degree. C. Periodically,
individual plaques from the assay were clonally isolated by
excision of the plaque area and placement of the excised agar plug
in a 96-well tray containing a monolayer of MDCK cells. The 96-well
trays were incubated overnight and the yield assayed for
temperature sensitivity by CPE assay in duplicate 96-well trays
incubated at about 34.degree. C. and at about 39.degree. C. The
percent of the clones that scored as temperature sensitive mutants
by this assay, i.e., the number of viral plaques that grew at
34.degree. C. but did not grow at 39.degree. C., divided by the
total number of plaques, was calculated, and is shown in Table 2.
Temperature sensitive isolates were then evaluated for protein
synthesis at the non-permissive temperature by visualization of
radiolabeled virus-synthesized proteins by SDS polyacrylamide gel
electrophoresis (SDS-PAGE).
2TABLE 2 Percent of isolated Clones that were temperature
sensitive. Percent Temperature Sensitive Passage # Limb A Limb B
Limb C Limb D Limb E p36 56% 66% 0% 66% 54% p46 80% 60% 75% p47 80%
p48 100% p49 100% 100% 50% p50 90% p51 100% p52 57% p62 100% 100%
p65 100% p66 100% 88%
[0066] From the clonal isolates tested for temperature sensitivity,
two were selected for further study. Clone EIV-P821 was selected
from the 49th passage of limb B and clone EIV-P824 was selected
from the 48th passage of limb C, as defined in Table 1. Both of
these virus isolates were temperature sensitive, with plaque
formation of both isolates inhibited at a temperature of about
39.degree. C. At this temperature, protein synthesis was completely
inhibited by EIV-P821, but EIV-P824 exhibited normal levels of
protein synthesis. In addition, plaque formation by EIV-P821 was
inhibited at a temperature of about 37.degree. C., and at this
temperature, late gene expression was inhibited, i.e., normal
levels of NP protein were synthesized, reduced or no M1 or HA
proteins were synthesized, and enhanced levels of the polymerase
proteins were synthesized. The phenotype observed at 37.degree. C.,
being typified by differential viral protein synthesis, was
distinct from the protein synthesis phenotype seen at about
39.degree. C., which was typified by the inhibition of synthesis of
all viral proteins. Virus EIV-P821 has been deposited with the
American Type Culture Collection (ATCC) under Accession No. ATCC
VR-2625, and virus EIV-P824 has been deposited with the ATCC under
Accession No. ATCC VR-2624.
[0067] C. Further characterization of the mutations in isolate
EIV-P821 were carried out by reassortment analysis, as follows.
Reassortment analysis in influenza viruses allows one skilled in
the art, under certain circumstances, to correlate phenotypes of a
given virus with putative mutations occurring on certain of the
eight RNA segments that comprise an influenza A virus genome. This
technique is described, for example, in Palese, et al., ibid. A
mixed infection of EIV-P821 and an avian influenza virus,
A/mallard/New York/6750/78 was performed as follows. MDCK cells
were co-infected with EIV-P821 at a multiplicity of infection (MOI)
of 2 pfu/cell and A/mallard/New York/6750/78 at an MOI of either 2,
5, or 10 pfu/cell. The infected cells were incubated at a
temperature of about 34.degree. C. The yields of the various
co-infections were titered and individual plaques were isolated at
about 34.degree. C., and the resultant clonal isolates were
characterized as to whether they were able to grow at about
39.degree. C. and about 37.degree. C., and express their genes,
i.e., synthesize viral proteins, at about 39.degree. C., about
37.degree. C., and about 34.degree. C. Protein synthesis was
evaluated by SDS-PAGE analysis of radiolabeled infected-cell
lysates. The HA, NP and NS-1 proteins of the two parent viruses,
each of which is encoded by a separate genome segment, were
distinguishable by SDS-PAGE analysis, since these particular viral
proteins, as derived from either the equine or the avian influenza
virus, migrate at different apparent molecular weights. In this way
it was possible, at least for the HA, NP, and NS-1 genes, to
evaluate whether certain phenotypes of the parent virus, e.g., the
temperature sensitive and the protein synthesis phenotypes,
co-segregate with the genome segments carrying these genes. The
results of the reassortment analyses investigating co-segregation
of a) the mutation inhibiting plaque formation, i.e., the induction
of CPE, at a non-permissive temperature of about 39.degree. C. or
b) the mutation inhibiting protein synthesis at a non-permissive
temperature of about 39.degree. C. with each of the EIV-P821 HA, NP
and NS-1 proteins are shown in Tables 3 and 4, respectively.
3TABLE 3 Reassortment analysis of the EIV-P821 39.degree. C. plaque
formation phenotype with avian influenza virus, A/mallard/New
York/6750/78 Gene Virus ts+.sup.1 ts-.sup.2 HA avian 26 13 equine
11 44 NP avian 37 8 equine 0 49 NS-1 avian 9 8 equine 12 20
.sup.1number of clonal isolates able to induce CPE in tissue
culture cells at a temperature of about 39.degree. C. .sup.2number
of clonal isolates inhibited in the ability to induce CPE in tissue
culture cells at a temperature of about 39.degree. C.
[0068]
4TABLE 4 Reassortment analysis of the EIV-P821 39.degree. C.
protein synthesis phenotype with avian influenza virus,
A/mallard/New York/6750/78 Gene Virus ts+.sup.1 ts-.sup.2 HA avian
18 1 equine 11 7 NP avian 34 5 equine 7 8 NS-1 avian 10 4 equine 14
5 .sup.1number of clonal isolate which synthesize all viral
proteins at a temperature of about 39.degree. C. .sup.2number of
clonal isolates inhibited in the ability to synthesize all viral
proteins at a temperature of about 39.degree. C.
[0069] The results demonstrated an association of the equine NP
gene with a mutation causing the inability of EIV-P821 to form
plaques at a non-permissive temperature of about 39.degree. C., but
the results did not suggest an association of any of the HA, NP, or
NS-1 genes with a mutation causing the inability of EIV-P821 to
express viral proteins at a non-permissive temperature of about
39.degree. C. Thus, these data also demonstrated that the plaque
formation phenotype and the protein synthesis phenotype observed in
virus EIV-P821 were the result of separate mutations.
[0070] D. Studies were also conducted to determine if cold-adapted
equine influenza viruses of the present invention have a dominant
interference phenotype, that is, whether they dominate in mixed
infection with the wild type parental virus A/Kentucky/1/91 (H3N8).
The dominant interference phenotype of viruses EIV-P821 and
EIV-P824 were evaluated in the following manner. Separate
monolayers of MDCK cells were singly infected with the parental
virus A/Kentucky/1/91 (H3N8) at an MOI of 2, singly infected with
either cold-adapted virus EIV-P821 or EIV-P824 at an MOI of 2, or
simultaneously doubly infected with both the parental virus and one
of the cold adapted viruses at an MOI of 2+2, all at a temperature
of about 34.degree. C. At 24 hours after infection, the media from
the cultures were harvested and the virus yields from the various
infected cells were measured by duplicate plaque assays performed
at temperatures of about 34.degree. C. and about 39.degree. C. This
assay took advantage of the fact that cold adapted equine influenza
viruses EIV-P821 or EIV-P824 are temperature sensitive and are thus
unable to form plaques at a non-permissive temperature of about
39.degree. C., while the parental virus is able to form plaques at
both temperatures, thus making it possible to measure the growth of
the parental virus in the presence of the cold adapted virus.
Specifically, the dominant interference effect of the cold adapted
virus on the growth of the parental virus was quantitated by
comparing the virus yield at about 39.degree. C. of the cells
singly infected with parental virus to the yield of the parental
virus in doubly infected cells. EIV-P821, in mixed infection, was
able to reduce the yield of the parental virus by approximately 200
fold, while EIV-P824, in mixed infection, reduced the yield of the
parental virus by approximately 3200 fold. This assay therefore
showed that cold-adapted equine influenza viruses EIV-P821 and
EIV-P824 both exhibit the dominant interference phenotype.
[0071] E. Virus isolate EIV-MSV+5 was derived from EIV-P821, as
follows. EIV-P821 was passaged once in eggs, as described above, to
produce a Master Seed Virus isolate, denoted herein as EIV-MSVO.
EIV-MSVO was then subjected to passage three additional times in
eggs, the virus isolates at the end of each passage being
designated EIV-MSV+1, EIW-MSV+2, and EIV-MSV+3, respectively.
EIV-MSV+3 was then subjected to two additional passages in MDCK
cells, as follows. MDCK cells were grown in 150 cm.sup.2 tissue
culture flasks in MEM tissue culture medium with Hanks Salts,
containing 10% calf serum. The cells were then washed with sterile
PBS and the growth medium was replaced with about 8 ml per flask of
infection medium (tissue culture medium comprising MEM with Hanks
Salts, 1 .mu.g/ml TPCK trypsin solution, 0.125% bovine serum
albumin (BSA), and 10 mM HEPES buffer). MDCK cells were inoculated
with AF containing virus EIV-MSV+3 (for the first passage in MDCK
cells) or virus stock harvested from EIV-MSV+4 (for the second
passage in MDCK cells), and the viruses were allowed to adsorb for
1 hour at about 34.degree. C. The inoculum was removed from the
cell monolayers, the cells were washed again with PBS, and about
100 ml of infection medium was added per flask. The infected cells
were incubated at about 34.degree. C. for 24 hours. The
virus-infected MDCK cells were harvested by shaking the flasks
vigorously to disrupt the cell monolayer, resulting in virus
isolates EIV-MSV+4 (the first passage in MDCK cells), and EIV-MSV+5
(the second passage in MDCK cells).
[0072] Viruses EIV-MSVO and EIV-MSV+5 were subjected to phenotypic
analysis, as described in section B above, to determine their
ability to form plaques and synthesize viral proteins at
temperatures of about 34.degree. C., about 37.degree. C., and about
39.degree. C. Both EIV-MSVO and EIV-MSV+5 formed plaques in tissue
culture cells at a temperature of about 34.degree. C., and neither
virus isolate formed plaques or exhibited detectable viral protein
synthesis at a temperature of about 39.degree. C. Virus EIV-MSVO
had a similar temperature sensitive phenotype as EIV-P821 at a
temperature of about 37.degree. C., i.e., it was inhibited in
plaque formation, and late gene expression was inhibited. However,
EIV-MSV+5, unlike its parent virus, EIV-P821, did form plaques in
tissue culture at a temperature of about 37.degree. C., and at this
temperature, the virus synthesized normal amounts of all proteins.
Virus EIV-MSV+5 has been deposited with the ATCC under Accession
No. ATCC VR-2627.
EXAMPLE 2
[0073] Therapeutic compositions of the present invention were
produced as follows.
[0074] A. A large stock of EIV-P821 was propagated in eggs as
follows. About 60 specific pathogen-free embryonated chicken eggs
were candled and non-viable eggs were discarded. Stock virus was
diluted to about 1.0.times.10.sup.5 pfu/ml in sterile PBS. Virus
was inoculated into the allantoic cavity of the eggs as described
in Example 1A. After a 3-day incubation in a humidified chamber at
a temperature of about 34.degree. C., AF was harvested from the
eggs according to the method described in Example 1A. The harvested
AF was mixed with a stabilizer solution, for example A1/A2
stabilizer, available from Diamond Animal Health, Des Moines, Iowa,
at 25% V/T (stabilizer/AF). The harvested AF was batched in a
centrifuge tube and was clarified by centrifugation for 10 minutes
at 1000 rpm in an IEC Centra-7R refrigerated table top centrifuge
fitted with a swinging bucket rotor. The clarified fluid was
distributed into 1-ml cryovials and was frozen at about -70.degree.
C. Virus stocks were titrated on MDCK cells by CPE and plaque assay
at about 34.degree. C.
[0075] B. A large stock of EIV-P821 was propagated in MDCK cells as
follows. MDCK cells were grown in 150 cm.sup.2 tissue culture
flasks in MEM tissue culture medium with Hanks Salts, containing
10% calf serum. The cells were then washed with sterile PBS and the
growth medium was replaced with about 8 ml per flask of infection
medium. The MDCK cells were inoculated with virus stock at an MOI
ranging from about 0.5 pfu per cell to about 0.005 pfu per cell,
and the viruses were allowed to adsorb for 1 hour at about
34.degree. C. The inoculum was removed from the cell monolayers,
the cells were washed again with PBS, and about 100 ml of infection
medium was added per flask. The infected cells were incubated at
about 34.degree. C. for 24 hours. The virus-infected MDCK cells
were harvested by shaking the flasks vigorously to disrupt the cell
monolayer and stabilizer solution was added to the flasks at 25%
V/V (stabilizer/virus solution). The supernatants were distributed
aseptically into cryovials and frozen at -70.degree. C.
[0076] C. Therapeutic compositions comprising certain cold-adapted
temperature sensitive equine influenza viruses of the present
invention were formulated as follows. Just prior to vaccination
procedures, such as those described in Examples 3-7 below, stock
vials of EIV-P821 or EIV-MSV+5 were thawed and were diluted in an
excipient comprising either water, PBS, or in MEM tissue culture
medium with Hanks Salts, containing 0.125% bovine serum albumin
(BSA-MEM solution) to the desired dilution for administration to
animals. The vaccine compositions were held on ice prior to
vaccinations. All therapeutic compositions were titered on MDCK
cells by standard methods just prior to vaccinations and wherever
possible, an amount of the composition, treated identically to
those administered to the animals, was titered after the
vaccinations to ensure that the virus remained viable during the
procedures.
EXAMPLE 3
[0077] A therapeutic composition comprising cold-adapted equine
influenza virus EIV-P821 was tested for safety and its ability to
replicate in three horses showing detectable prior immunity to
equine influenza virus as follows. EIV-P821, produced as described
in Example 1A, was grown in eggs as described in Example 2A and was
formulated into a therapeutic composition comprising 10.sup.7 pfu
EIV-P821/2 ml BSA-MEM solution as described in Example 2C.
[0078] Three ponies having prior detectable hemagglutination
inhibition (HAI) titers to equine influenza virus were inoculated
with a therapeutic composition comprising EIV-P821 by the following
method. Each pony was given a 2-ml dose of EIV-P821, administered
intranasally using a syringe fitted with a blunt cannula long
enough to reach past the false nostril, 1 ml per nostril.
[0079] The ponies were observed for approximately 30 minutes
immediately following and at approximately four hours after
vaccination for immediate type allergic reactions such as sneezing,
salivation, labored or irregular breathing, shaking, anaphylaxis,
or fever. The animals were further monitored on days 1-11
post-vaccination for delayed type allergic reactions, such as
lethargy or anorexia. None of the three ponies in this study
exhibited any allergic reactions from the vaccination.
[0080] The ponies were observed daily, at approximately the same
time each day, starting two days before vaccination and continuing
through day 11 following vaccination for clinical signs consistent
with equine influenza. The ponies were observed for nasal
discharge, ocular discharge, anorexia, disposition, heart rate,
capillary refill time, respiratory rate, dyspnea, coughing, lung
sounds, presence of toxic line on upper gum, and body temperature.
In addition submandibular and parietal lymph nodes were palpated
and any abnormalities were described. None of the three ponies in
this study exhibited any abnormal reactions or overt clinical signs
during the observation period.
[0081] To test for viral shedding in the animals, on days 0 through
11 following vaccination, nasopharyngeal swabs were collected from
the ponies as described in Chambers, et al., 1995, Equine Practice,
17, 19-23. Chambers, et al., ibid., is incorporated herein by
reference in its entirety. Briefly, two sterile Dacron polyester
tipped applicators (available, e.g., from Hardwood Products Co.,
Guilford, Me.) were inserted, together, into each nostril of the
ponies. The swabs (four total, two for each nostril) were broken
off into a 15-ml conical centrifuge tube containing 2.5 ml of
chilled transport medium comprising 5% glycerol, penicillin,
streptomycin, neomycin, and gentamycin in PBS at physiological pH.
Keeping the samples on wet ice, the swabs were aseptically wrung
out into the medium and the nasopharyngeal samples were divided
into two aliquots. One aliquot was used to attempt isolation of EIV
by inoculation of embryonated eggs, using the method described in
Example 1. The AF of the inoculated eggs was then tested for its
ability to cause hemagglutination, by standard methods, indicating
the presence of equine influenza virus in the AF. On days 2 and 3
post-vaccination, the other aliquots were tested for virus by the
Directigen.RTM. Flu A test, available from Becton-Dickinson
(Cockeysville, Md.).
[0082] Attempts to isolate EIV from the nasopharyngeal secretions
of the three animals by egg inoculation were unsuccessful. However
on days 2 and 3, all animals tested positive for the presence of
virus shedding using the Directigen Flu A test, consistent with the
hypothesis that EIV-P821 was replicating in the seropositive
ponies.
[0083] To test the antibody titers to EIV in the inoculated animals
described in this example, as well as in the animals described in
Examples 4-7, blood was collected from the animals prior to
vaccination and on designated days post-vaccination. Serum was
isolated and was treated either with trypsin/periodate or kaolin to
block the nonspecific inhibitors of hemagglutination present in
normal sera. Serum samples were tested for hemagglutination
inhibition (HAI) titers against a recent EIV isolate by standard
methods, described, for example in the "Supplemental assay method
for conducting the hemagglutination inhibition assay for equine
influenza virus antibody" (SAM 124), provided by the U.S.D.A.
National Veterinary Services Laboratory under 9 CFR 113.2, which is
incorporated by reference herein in its entirety.
[0084] The HAI titers of the three ponies are shown in Table 5. As
can be seen, regardless of the initial titer, the serum HAI titers
increased at least four-fold in all three animals after vaccination
with EIV-P821.
[0085] These data demonstrate that cold-adapted equine influenza
virus EIV-P821 is safe and non-reactogenic in sero-positive ponies,
and that these animals exhibited an increase in antibody titer to
equine influenza virus, even though they had prior demonstrable
titers.
5TABLE 5 HAI titers of vaccinated animals* Animal HAI Titer (days
after vaccination) ID 0 7 14 21 18 40 80 160 160 19 10 20 40 80 25
20 40 320 80 *HAI titers are expressed as the reciprocal of the
highest dilution of serum which inhibited hemagglutination of
erythrocytes by a recent isolate of equine influenza virus.
EXAMPLE 4
[0086] This Example discloses an animal study to evaluate the
safety and efficacy of a therapeutic composition comprising
cold-adapted equine influenza virus EIV-P821.
[0087] A therapeutic composition comprising cold-adapted equine
influenza virus EIV-P821 was tested for attenuation, as well as its
ability to protect horses from challenge with virulent equine
influenza virus, as follows. EIV-P821, produced as described in
Example 1, was grown in eggs as described in Example 2A and was
formulated into a therapeutic composition comprising 10.sup.7 pfu
of virus/2 ml water, as described in Example 2C. Eight
EIV-seronegative ponies were used in this study. Three of the eight
ponies were vaccinated with a 2-ml dose comprising 10.sup.7 pfu of
the EIV-P821 therapeutic composition, administered intranasally,
using methods similar to those described in Example 3. One pony was
given 10.sup.7 pfu of the EIV-P821 therapeutic composition,
administered orally, by injecting 6 ml of virus into the pharynx,
using a 10-ml syringe which was adapted to create a fine spray by
the following method. The protruding "seat" for the attachment of
needles was sealed off using modeling clay and its cap was left in
place. About 10 holes were punched through the bottom of the
syringe, i.e., surrounding the "seat," using a 25-gauge needle. The
syringe was placed into the interdental space and the virus was
forcefully injected into the back of the mouth. The remaining four
ponies were held as non-vaccinated controls.
[0088] The vaccinated ponies were observed for approximately 30
minutes immediately following and at approximately four hours after
vaccination for immediate type allergic reactions, and the animals
were further monitored on days 1-11 post-vaccination for delayed
type allergic reactions, both as described in Example 3. None of
the four vaccinated ponies in this study exhibited any abnormal
reactions from the vaccination.
[0089] The ponies were observed daily, at approximately the same
time each day, starting two days before virus vaccination and
continuing through day 11 following vaccination for clinical signs,
such as those described in Example 3. None of the four vaccinated
ponies in this study exhibited any clinical signs during the
observation period. This result demonstrated that cold-adapted
equine influenza virus EIV-P821 exhibits the phenotype of
attenuation.
[0090] To test for viral shedding in the vaccinated animals, on
days 0 through 11 following vaccination, nasopharyngeal swabs were
collected from the ponies as described in Example 3. The
nasopharyngeal samples were tested for virus in embryonated chicken
eggs according to the method described in Example 3.
[0091] As shown in Table 6, virus was isolated from only one
vaccinated animal using the egg method. However, as noted in
Example 3, the lack of isolation by this method does not preclude
the fact that virus replication is taking place, since replication
may be detected by more sensitive methods, e.g., the Directigen Flu
A test.
6TABLE 6 Virus isolation in eggs after vaccination Animal Virus
Isolation (days after vaccination) ID Route 0 1 2 3 4 5 6 7 8 9 10
11 91 IN - - + + + + + + + + + - 666 IN - - - - - - - - - - - - 673
IN - - - - - - - - - - - - 674 Oral - - - - - - - - - - - -
[0092] To test the antibody titers to equine influenza virus in the
vaccinated animals, blood was collected from the animals prior to
vaccination and on days 7, 14, 21, and 28 post-vaccination. Serum
samples were isolated and were tested for hemagglutination
inhibition (HAI) titers against a recent EIV isolate according to
the methods described in Example 3.
[0093] The HAI titers of the four vaccinated ponies are shown in
Table 7.
7TABLE 7 HAI titers after vaccination Animal HAI Titer (days after
vaccination) ID Route 0 7 14 21 28 91 IN <10 <10 <10
<10 <10 666 IN 10 10 10 20 20 673 IN 10 10 10 20 20 674 Oral
20 40 40 40 40
[0094] Unlike the increase in HAI titer observed with the three
animals described in the study in Example 3, the animals in this
study did not exhibit a significant increase, i.e., greater than
four-fold, in HAI titer following vaccination with EIV-P821.
[0095] Approximately four and one-half months after vaccine virus
administration, all 8 ponies, i.e., the four that were vaccinated
and the four non-vaccinated controls, were challenged by the
following method. For each animal, 10.sup.7 pfu of the virulent
equine influenza virus strain A/equine/Kentucky/1/91 (H3N8) was
suspended in 5 ml of water. A mask was connected to a nebulizer,
and the mask was placed over the animal's muzzle, including the
nostrils. Five (5) ml was nebulized for each animal, using settings
such that it took 5-10 minutes to deliver the full 5 ml. Clinical
observations, as described in Example 3, were performed on all
animals three days before challenge and daily for 11 days after
challenge.
[0096] Despite the fact that the vaccinated animals did not exhibit
marked increases in their HAI titers to equine influenza virus, all
four vaccinated animals were protected against equine influenza
virus challenge. None of the vaccinated animals showed overt
clinical signs or fever, although one of the animals had a minor
wheeze for two days. On the other hand, all four non-vaccinated
ponies shed virus and developed clinical signs and fever typical of
equine influenza virus infection. Thus, this example demonstrates
that a therapeutic composition of the present invention can protect
horses from equine influenza disease.
EXAMPLE 5
[0097] This Example discloses an additional animal study to
evaluate attenuation of a therapeutic composition comprising
cold-adapted equine influenza virus EIV-P821, and its ability to
protect vaccinated horses from subsequent challenge with virulent
equine influenza virus. Furthermore, this study evaluated the
effect of exercise stress on the safety and efficacy of the
therapeutic composition.
[0098] A therapeutic composition comprising cold-adapted equine
influenza virus EIV-P821 was tested for safety and efficacy in
horses, as follows. EIV-P821, produced as described in Example 1,
was grown in eggs as described in Example 2A and was formulated
into a therapeutic composition comprising 10.sup.7 pfu virus/5 ml
water, as described in Example 2C. Fifteen ponies were used in this
study. The ponies were randomly assigned to three groups of five
animals each, as shown in Table 8, there being two vaccinated
groups and one unvaccinated control group. The ponies in group 2
were exercise stressed before vaccination, while the ponies in
vaccinate group 1 were held in a stall.
8TABLE 8 Vaccination/challenge protocol Group No. Ponies Exercise
Vaccine Challenge 1 5 -- Day 0 Day 90 2 5 Days -4 to 0 Day 0 Day 90
3 5 -- -- Day 90
[0099] The ponies in group 2 were subjected to exercise stress on a
treadmill prior to vaccination, as follows. The ponies were
acclimated to the use of the treadmill by 6 hours of treadmill use
at a walk only. The actual exercise stress involved a daily
exercise regimen starting 4 days before and ending on the day of
vaccination (immediately prior to vaccination). The treadmill
exercise regimen is shown in Table 9.
9TABLE 9 Exercise regimen for the ponies in Group 2 Speed (m/sec)
Time (min.) Incline (.degree.) 1.5 2 0 3.5 2 0 3.5 2 7 4.5.dagger.
2 7 5.5.dagger. 2 7 6.5.dagger. 2 7 7.5.dagger. 2 7 8.5.dagger. 2 7
3.5 2 7 1.5 10 0.dagger. .dagger.Speed, in meters per second
(m/sec) was increased for each animal every 2 minutes until the
heart rate reached and maintained .gtoreq.200 beats per minute
[0100] Groups 1 and 2 were given a therapeutic composition
comprising 10.sup.7 pfu of EIV-P821, by the nebulization method
described for the challenge described in Example 4. None of the
vaccinated ponies in this study exhibited any immediate or delayed
allergic reactions from the vaccination.
[0101] The ponies were observed daily, at approximately the same
time each day, starting two days before vaccination and continuing
through day 11 following vaccination for clinical signs, such as
those described in Example 3. None of the vaccinated ponies in this
study exhibited any overt clinical signs during the observation
period.
[0102] To test for viral shedding in the vaccinated animals, before
vaccination and on days 1 through 11 following vaccination,
nasopharyngeal swabs were collected from the ponies as described in
Example 3. The nasopharyngeal samples were tested for virus in
embryonated chicken eggs according to the method described in
Example 3. Virus was isolated from the vaccinated animals, i.e.,
Groups 1 and 2, as shown in Table 10.
10TABLE 10 Virus isolation after vaccination Animal Virus Isolation
(days after vaccination) Group ID Exercise 0 1 2 3 4 5 6 7 8 9 10
11 1 12 No - - + + + + + - + + - - 16 - - + + + + + - - - - - 17 -
- + + + + + + + - + - 165 - - - - - - - - - - - - 688 - - - - - + -
+ - - - - 2 7 Yes - - - + + + + - - - - - 44 - - - - - - - - - - -
- 435 - - + + + + - - - - - - 907 - - - + - + + - - - - - 968 - - -
- - + - + - - - -
[0103] To test the antibody titers to equine influenza virus in the
vaccinated animals, blood was collected prior to vaccination and on
days 7, 14, 21, and 28 post-vaccination. Serum samples were
isolated and were tested for HAI titers against a recent EIV
isolate according to the methods described in Example 3. These
titers are shown in Table 11.
11TABLE 11 HAI titers after vaccination and after challenge on day
90 Animal Day Post-vaccination Group ID -1 7 14 21 28 91 105 112
119 126 1 12 <10 <10 <10 <10 <10 <10 80 320 320
640 1 16 <10 <10 20 20 <10 <10 20 160 320 320 1 17
<10 <10 10 10 10 10 80 160 160 160 1 165 <10 <10 10 10
10 10 80 80 80 80 1 688 <10 <10 20 20 20 20 20 20 20 40 2 7
<10 <10 10 10 <10 <10 20 80 80 40 2 44 <10 <10 20
20 20 10 80 320 320 320 2 435 <10 <10 20 20 10 <10 20 80
80 80 2 907 <10 <10 10 10 20 10 10 40 80 80 2 968 <10
<10 <10 <10 <10 <10 40 160 160 160 3 2 <10 80 640
640 320 3 56 <10 80 320 320 320 3 196 <10 20 160 80 80 3 198
10 40 160 320 320 3 200 <10 20 80 80 40 Group Description 1
Vaccination only 2 Vaccination and Exercise 3 Control
[0104] On day 90 post vaccination, all 15 ponies were challenged
with 10.sup.7 pfu of equine influenza virus strain
A/equine/Kentucky/1/91 (H3N8) by the nebulizer method as described
in Example 4. Clinical observations, as described in Example 3,
were performed on all animals three days before challenge and daily
for 11 days after challenge. There were no overt clinical signs
observed in any of the vaccinated ponies. Four of the five
non-vaccinated ponies developed fever and clinical signs typical of
equine influenza virus infection.
[0105] Thus, this example demonstrates that a therapeutic
composition of the present invention protects horses against equine
influenza disease, even if the animals are stressed prior to
vaccination.
EXAMPLE 6
[0106] This Example compared the infectivities of therapeutic
compositions of the present invention grown in eggs and grown in
tissue culture cells. From a production standpoint, there is an
advantage to growing therapeutic compositions of the present
invention in tissue culture rather than in embryonated chicken
eggs. Equine influenza virus, however, does not grow to as high a
titer in cells as in eggs. In addition, the hemagglutinin of the
virus requires an extracellular proteolytic cleavage by
trypsin-like proteases for infectivity. Since serum contains
trypsin inhibitors, virus grown in cell culture must be propagated
in serum-free medium that contains trypsin in order to be
infectious. It is well known by those skilled in the art that such
conditions are less than optimal for the viability of tissue
culture cells. In addition, these growth conditions may select for
virus with altered binding affinity for equine cells, which may
affect viral infectivity since the virus needs to bind efficiently
to the animal's nasal mucosa to replicate and to stimulate
immunity. Thus, the objective of the study disclosed in this
example was to evaluate whether the infectivity of therapeutic
compositions of the present invention was adversely affected by
growth for multiple passages in in vitro tissue culture.
[0107] EIV-P821, produced as described in Example 1, was grown in
eggs as described in Example 2A or in MDCK cells as described in
Example 2B. In each instance, the virus was passaged five times.
EIV-P821 was tested for its cold-adaptation and temperature
sensitive phenotypes after each passage. The egg and cell-passaged
virus preparations were formulated into therapeutic compositions
comprising 10.sup.7 pfu virus/2 ml BSA-MEM solution, as described
in Example 2C, resulting in an egg-grown EIV-P821 therapeutic
composition and an MDCK cell-grown EIV-P821 therapeutic
composition, respectively.
[0108] Eight ponies were used in this study. Serum from each of the
animals was tested for HAI titers to equine influenza virus prior
to the study. The animals were randomly assigned into one of two
groups of four ponies each. Group A received the egg-grown EIV-P821
therapeutic composition, and Group B received the MDCK-grown
EIV-P821 therapeutic composition, prepared as described in Example
2B. The therapeutic compositions were administered intranasally by
the method described in Example 3.
[0109] The ponies were observed daily, at approximately the same
time each day, starting two days before vaccination and continuing
through day 11 following vaccination for allergic reactions or
clinical signs as described in Example 3. No allergic reactions or
overt clinical signs were observed in any of the animals.
[0110] Nasopharyngeal swabs were collected before vaccination and
daily for 11 days after vaccination. The presence of virus material
in the nasal swabs was determined by the detection of CPE on MDCK
cells infected as described in Example 1, or by inoculation into
eggs and examination of the ability of the infected AF to cause
hemagglutination, as described in Example 3. The material was
tested for the presence of virus only, and not for titer of virus
in the sample. Virus isolation results are listed in Table 12.
Blood was collected and serum samples from days 0, 7, 14, 21 and 28
after vaccination were tested for hemagglutination inhibition
antibody titer against a recent isolate. HAI titers are also listed
in Table 12.
12TABLE 12 HAI titers and virus isolation after vaccination HAI
Titer (DPV.sup.3) Virus Isolation.sup.1 (DPV.sup.3) Group.sup.2 ID
0 7 14 21 28 0 1 2 3 4 5 6 7 8 9 10 11 1 31 <10 20 160 160 160
-- EC -- C EC EC C C EC -- -- -- 37 <10 40 160 160 160 -- EC C C
EC C C C -- -- -- -- 40 <10 20 80 160 80 -- EC EC C -- C EC C --
EC EC -- 41 <10 40 160 160 80 -- EC EC C EC C EC EC -- -- -- --
2 32 <10 <10 80 80 40 -- EC -- C -- C -- C -- EC -- -- 34
<10 20 160 160 160 -- EC -- C EC C EC C -- -- -- -- 35 <10
<10 80 80 40 -- EC -- C -- C -- C -- EC -- -- 42 <10 <10
80 80 40 -- -- -- C -- C EC EC -- -- -- -- .sup.1E = Egg isolation
positive; C = CPE isolation positive; -- = virus not detected by
either of the methods .sup.2Group 1: Virus passaged 5X in MDCK
cells; Group 2: Virus passaged 5X in Eggs .sup.3Days
Post-vaccination
[0111] The results in Table 12 show that there were no significant
differences in infectivity or immunogenicity between the egg-grown
and MDCK-grown EIV-P821 therapeutic compositions.
EXAMPLE 7
[0112] This example evaluated the minimum dose of a therapeutic
composition comprising a cold-adapted equine influenza virus
required to protect a horse from equine influenza virus
infection.
[0113] The animal studies disclosed in Examples 3-6 indicated that
a therapeutic composition of the present invention was efficacious
and safe. In those studies, a dose of 10.sup.7 pfu, which
correlates to approximately 10.sup.8 TCID.sub.50 units, was used.
However, from the standpoints of cost and safety, it is
advantageous to use the minimum virus titer that will protect a
horse from disease caused by equine influenza virus. In this study,
ponies were vaccinated with four different doses of a therapeutic
composition comprising a cold-adapted equine influenza virus to
determine the minimum dose which protects a horse against virulent
equine influenza virus challenge.
[0114] EIV-P821, produced as described in Example 1A, was passaged
and grown in MDCK cells as described in Example 2B and was
formulated into a therapeutic composition comprising either
2.times.10.sup.4, 2.times.10.sup.5, 2.times.10.sup.6, or
2.times.10.sup.7 TCID.sub.50 units/1 ml BSA-MEM solution as
described in Example 2C. Nineteen horses of various ages and breeds
were used for this study. The horses were assigned to four vaccine
groups, one group of three horses and three groups of four horses,
and one control group of four horses (see Table 13). Each of the
ponies in the vaccine groups were given a 1-ml dose of the
indicated therapeutic composition, administered intranasally by
methods similar to those described in Example 3.
13TABLE 13 Vaccination protocol Vaccine Dose, Group No. No. Animals
TCID.sub.50 Units 1 3 2 .times. 10.sup.7 2 4 2 .times. 10.sup.6 3 4
2 .times. 10.sup.5 4 4 2 .times. 10.sup.4 5 4 control
[0115] The ponies were observed for approximately 30 minutes
immediately following and at approximately four hours after
vaccination for immediate type reactions, and the animals were
further monitored on days 1-11 post-vaccination for delayed type
reactions, both as described in Example 3. None of the vaccinated
ponies in this study exhibited any abnormal reactions or overt
clinical signs from the vaccination.
[0116] Blood for serum analysis was collected 3 days before
vaccination, on days 7, 14, 21, and 28 after vaccination, and after
challenge on Days 35 and 42. Serum samples were tested for HAI
titers against a recent EIV isolate according to the methods
described in Example 3. These titers are shown in Table 14. Prior
to challenge on day 29, 2 of the 3 animals in group 1, 4 of the 4
animals in group 2, 3 of the 4 animals in group 3, and 2 of the 4
animals in group 4 showed at least 4-fold increases in HAI titers
after vaccination. In addition, 2 of the 4 control horses also
exhibited increases in HAI titers. One interpretation for this
result is that the control horses were exposed to vaccine virus
transmitted from the vaccinated horses, since all the horses in
this study were housed in the same barn.
14TABLE 14 HAI titers post-vaccination and post-challenge, and
challenge results Dose in Animal Vaccination on Day 0, Challenge on
Day 29 Chall. Sick No. TCID.sub.50 units ID -1 7 14 21 28 35 42 +/-
1 2 .times. 10.sup.7 41 <10 <10 10 40 10 20 80 - 42 40 40 40
40 40 <10 80 - 200 <10 <10 80 40 160 40 40 - 2 2 .times.
10.sup.6 679 <10 10 40 40 40 20 20 - 682 <10 <10 40 40 40
40 40 - 795 20 80 160 160 320 320 640 - R <10 10 40 20 160 40 40
- 3 2 .times. 10.sup.5 73 <10 <10 160 40 80 160 160 - 712
<10 <10 20 20 40 40 20 - 720 <10 20 80 40 80 80 160 - 796
<10 <10 <10 <10 <10 10 80 + 4 2 .times. 10.sup.4 75
<10 <10 <10 <10 <10 <10 160 + 724 <10 >10
<10 <10 <10 20 320 + 789 <10 10 320 160 320 320 320 -
790 <10 <10 80 40 160 80 40 5 Control 12 <10 <10 <10
20 20 40 40 - 22 10 20 40 10 160 40 640 - 71 <10 <10 <10
<10 10 20 160 + 74 <10 <10 <10 <10 <10 <10 20
+
[0117] On day 29 post vaccination, all 19 ponies were challenged
with equine influenza virus strain A/equine/Kentucky/1/91 (H3N8) by
the nebulizer method as described in Example 4. The challenge dose
was prospectively calculated to contain about 10.sup.8 TCID.sub.50
units of challenge virus in a volume of 5 ml for each animal.
Clinical observations, as described in Example 3, were monitored
beginning two days before challenge, the day of challenge, and for
11 days following challenge. As shown in Table 14, no animals in
groups 1 or 2 exhibited clinical signs indicative of equine
influenza disease, and only one out of four animals in group 3
became sick. Two out of four animals in group 4 became sick, and
only two of the four control animals became sick. The results in
Table 14 suggest a correlation between seroconversion and
protection from disease, since, for example, the two control
animals showing increased HAI titers during the vaccination period
did not show clinical signs of equine influenza disease following
challenge. Another interpretation, however, was that the actual
titer of the challenge virus may have been less than the calculated
amount of 10.sup.8 TCID.sub.50 units, since, based on prior
results, this level of challenge should have caused disease in all
the control animals.
[0118] Nonetheless, the levels of seroconversion and the lack of
clinical signs in the groups that received a therapeutic
composition comprising at least 2.times.10.sup.6 TCID.sub.50 units
of a cold-adapted equine influenza virus suggests that this amount
was sufficient to protect a horse against equine influenza disease.
Furthermore, a dose of 2.times.10.sup.5 TCID.sub.50 units induced
seroconversion and gave clinical protection from challenge in 3 out
of 4 horses, and thus even this amount may be sufficient to confer
significant protection in horses against equine influenza
disease.
EXAMPLE 8
[0119] This example discloses reports of a non-specific
interference effect of a therapeutic composition comprising
cold-adapted equine influenza virus EIV-P821.
[0120] A therapeutic composition comprising cold-adapted equine
influenza virus EIV-P821, produced as described in Example 1, was
grown in eggs similarly to the procedure described in Example 2A,
was expanded by passage in MDCK cells similarly to the procedure
described in Example 2B, and was formulated into a therapeutic
composition as described in Example 2C. Thirty-four horses on 3
farms were involved in these observations. All the horses had a
nasal discharge from undetermined causes. Two horses about 1 to 2
years old on farm 1, 15 horses about 1 to 2 years old on farm 2 and
8 foals about age 3 to 6 months on farm 3 were each administered a
therapeutic composition produced as described in Example 2,
intranasally into one nostril using a syringe with a delivery
device tip attached to the end, with a 1.0 ml dose comprising about
10.sup.7.5 TCID.sub.50 of the therapeutic composition. On farm 3, 9
foals with nasal discharge did not receive a therapeutic
composition of the present invention.
[0121] On farm 1, the nasal discharge in the 2 horses improved by 2
days after administration of the therapeutic composition. On farm
2, the nasal discharge in the 15 horses improved by 1 day after
administration of the therapeutic composition. On farm 3, the nasal
discharge improved in 7 of the 8 vaccinated foals by 2 days after
administration of the therapeutic composition. The nasal discharge
in the 9 foals that were not administered the therapeutic
composition did not improve.
EXAMPLE 9
[0122] This example discloses a report of an antiviral effect of a
therapeutic composition comprising cold-adapted equine influenza
virus EIV-P821.
[0123] A therapeutic composition comprising cold-adapted equine
influenza virus EIV-P821, produced as described in Example 1, was
grown in eggs similarly to the procedure described in Example 2A,
was expanded by passage in MDCK cells similarly to the procedure
described in Example 2B, and was formulated into a therapeutic
composition as described in Example 2C. One hundred horses, all of
whom were showing clinical signs of influenza, were administered
the therapeutic composition in a manner as described in Example 8.
At the time of administration, about 20 to 30 horses were in the
early stage of influenza (with serous nasal discharge and high
temperature) and about 70 to 80 horses were in a later stage of
influenza (mucopurulent nasal discharge, most with no fever). The
veterinarian also treated about 30 of the horses in the later stage
of influenza with antibiotic. The clinical signs of infection
cleared within 3 days of treatment in 90 of the horses (including
all the horses in the early stage of influenza, none of which were
given antibiotics). The other 10 horses were continued on
antibiotic and clinical signs of infection were cleared in a week.
All the horses were back in race training within 10 days of
initiation of treatment. Prior to therapeutic compositions of the
present invention, veterinarians treated sick horses with
antibiotics alone, and it took at least 2 weeks for the horses to
clear the clinical signs of infection and at least 3 weeks before
the horses were back in race training In fact, in about 50% of the
cases the veterinarian would have to switch antibiotics and
continue to treat the horses.
[0124] While various embodiments of the present invention have been
described in detail, it is apparent that modifications and
adaptations of those embodiments will occur to those skilled in the
art. It is to be expressly understood, however, that such
modifications and adaptations are within the scope of the present
invention, as set forth in the following claims.
* * * * *